Comparative study of endophytic and endophytic diazotrophic bacterial communities across rice landraces grown in the highlands of northern Thailand

Communities of bacterial endophytes within the rice landraces cultivated in the highlands of northern Thailand were studied using fingerprinting data of 16S rRNA and nifH genes profiling by polymerase chain reaction–denaturing gradient gel electrophoresis. The bacterial communities’ richness, diversity index, evenness, and stability were varied depending on the plant tissues, stages of growth, and rice cultivars. These indices for the endophytic diazotrophic bacteria within the landrace rice Bue Wah Bo were significantly the lowest. The endophytic bacteria revealed greater diversity by cluster analysis with seven clusters compared to the endophytic diazotrophic bacteria (three clusters). Principal component analysis suggested that the endophytic bacteria showed that the community structures across the rice landraces had a higher stability than those of the endophytic diazotrophic bacteria. Uncultured bacteria were found dominantly in both bacterial communities, while higher generic varieties were observed in the endophytic diazotrophic bacterial community. These differences in bacterial communities might be influenced either by genetic variation in the rice landraces or the rice cultivation system, where the nitrogen input affects the endophytic diazotrophic bacterial community

Growth, symbiotic, and proteomics studies of soybean Bradyrhizobium in response to adaptive acid tolerance

Bradyrhizobial inoculated soybean often performs poorly on acid-soil because of the acid sensitivity of their associated root nodule bacteria. Acid tolerance in rhizobia has been considered as a key phenotypic characteristic in that it enables the bacteria to perform well under the restrictive conditions of excessive acidity. Since bacteria could develop acid tolerance to a more acid condition by using adaptive acid tolerance response (ATR), it is interesting to investigate whether bradyrhizobia could have this response and what proteins are involved in ATR. Bradyrhizobium sp. DASA01007 was selected for this study based on its ATR ability and symbiosis efficiency with soybean under acid condition. To establish an ATR in bradyrhizobia, late log phase culture of cell grown in mild acid condition was subsequently used as inoculum to more acid conditions. The 2D-gel and proteomic analyses were used to investigate the proteins response during ATR compared with non-adaptive conditions. The 29 identified proteins were grouped into 8 categories based on category orthologous group (COG) and one group of unknown categories. Hypothetical protein, transport and binding proteins, and translation protein were up-regulated at pH 4.5N (non-adaptive condition). While up-regulated proteins found during growth at pH 4.5A (adaptive condition) consisted of proteins in cellular processes, translation, energy metabolism, regulatory functions, interconversions and salvage of nucleosides and nucleotides, and conserved hypothetical proteins group. However, transport and binding proteins were absent in adaptive condition. At pH 5.5A, proteins involved in cellular processes were also detected. Several proteins overproduced in adaptive condition may be involved in ATR of bradyrhizobia. An importance of ATR in root nodule bacteria would support a better chance of survival in low pH soils than those conventionally grown in neutral pH. These results suggest that the use of ATR condition could provide an improvement in the production of inoculants.Keywords: Adaptive acid tolerance, Bradyrhizobium, Soybean, 2D-gel electrophoresi

Co-inoculation effects of Bradyrhizobium japonicum and Azospirillum sp. on competitive nodulation and rhizosphere eubacterial community structures of soybean under rhizobia-established soil conditions

Bradyrhizobial inoculants used for soybean seed inoculation to maximize the benefit of N2-fixation should include bradyrhizobial strain with high N2-fixation rates and ability to compete with the indigenous rhizobial populations. In this study, co-inoculation of plant growth promoting rhizobacteria (PGPR) Azospirillum sp. with either of Bradyrhizobium japonicum CB 1809 or USDA 110 increased shoot and root dry weight of soybean over non-inoculated control under pot condition with no indigenous soybean nodulating bradyrhizobia. Moreover, competition for nodulation and the effects on rhizosphere soil eubacterial community structures by using single or co-inoculation of B. japonicum and Azospirillum sp. under rhizobia-established Myanmar and Thailand soils were investigated. By inoculation of gus-marked USDA 110 singly or its co-inoculation gave 93.21 to 94.75% and 74.21 to 100% in nodule occupancy, and 23.50 to 41.95% and 50.37 to 73.24% promotion in biomass dry weight over non-inoculated control in Myanmar and Thailand soil samples, respectively. Each of all the tested inoculum levels, that is 106, 107 and 108 cfu/ml of Azospirillum sp. enhanced nodulation in combination with USDA 110 with a corresponding increase in 73.8, 62.25 and 95.34%; and 51.52, 62.38 and 79.46% over non-inoculated control, respectively in Myanmar and Thailand soil, respectively. In addition, soybean rhizosphere soil eubacterial community structures were not shifted by bacterial inoculation. Therefore, Azospirillum sp. could be used in co-inoculant production with B. japonicum for soybean.Keywords: Bradyrhizobium, plant growth promoting rhizobacteria (PGPR), soybean, co-inoculation, competition, rhizosphere eubacterial community structureAfrican Journal of Biotechnology Vol. 12(20), pp. 2850-286

Rhizobacteria and arbuscular mycorrhizal fungi of oil crops (physic nut and sacha inchi): a cultivable-based assessment for abundance, diversity, and plant growth-promoting potentials

Nowadays, oil crops are very attractive both for human consumption and biodiesel production; however, little is known about their commensal rhizosphere microbes. In this study, rhizosphere samples were collected from physic nut and sacha inchi plants grown in several areas of Thailand. Rhizobacteria, cultivable in nitrogen-free media, and arbuscular mycorrhizal (AM) fungi were isolated and examined for abundance, diversity, and plant growth-promoting activities (indole-3-acetic acid (IAA) and siderophore production, nitrogen fixation, and phosphate solubilization). Results showed that only the AM spore amount was affected by plant species and soil features. Considering rhizobacterial diversity, two classes—Alphaproteobacteria (Ensifer sp. and Agrobacterium sp.) and Gammaproteobacteria (Raoultella sp. and Pseudomonas spp.)—were identified in physic nut rhizosphere, and three classes; Actinobacteria (Microbacterium sp.), Betaproteobacteria (Burkholderia sp.) and Gammaproteobacteria (Pantoea sp.) were identified in the sacha inchi rhizosphere. Considering AM fungal diversity, four genera were identified (Acaulospora, Claroideoglomus, Glomus, and Funneliformis) in sacha inchi rhizospheres and two genera (Acaulospora and Glomus) in physic nut rhizospheres. The rhizobacteria with the highest IAA production and AM spores with the highest root-colonizing ability were identified, and the best ones (Ensifer sp. CM1-RB003 and Acaulospora sp. CM2-AMA3 for physic nut, and Pantoea sp. CR1-RB056 and Funneliformis sp. CR2-AMF1 for sacha inchi) were evaluated in pot experiments alone and in a consortium in comparison with a non-inoculated control. The microbial treatments increased the length and the diameter of stems and the chlorophyll content in both the crops. CM1-RB003 and CR1-RB056 also increased the number of leaves in sacha inchi. Interestingly, in physic nut, the consortium increased AM fungal root colonization and the numbers of offspring AM spores in comparison with those observed in sacha inchi. Our findings proved that AM fungal abundance and diversity likely rely on plant species and soil features. In addition, pot experiments showed that rhizosphere microorganisms were the key players in the development and growth of physic nut and sacha inchi

Type 3 Secretion System (T3SS) of Bradyrhizobium sp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association.

The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice

Selection and evaluation of Bradyrhizobium inoculum for peanut, Arachis hypogea production in the Lao People’s Democratic Republic

The interaction between leguminous plants and Bradyrhizobium is limited, known as host specificity. Therefore, the selection of an appropriate Bradyrhizobia for use as biofertilizer inoculum for legumes is necessary. The Arachis hypogea L. is the most popular legume produced in the Lao People's Democratic Republic (PDR). Therefore, this research aimed to obtain the appropriate Bradyrhizobia that provides high efficiency in A. hypogea production in the Lao PDR. The 14 isolates were obtained from root nodules of A. hypogea L. trapped with Lao PDR soil samples. Three were the top isolates PMVTL-01, SMVTL-02, and BLXBL-03 showing high efficiency for peanut growth promotion. Strains PMVTL-01 and SMVTL-02 were closely related to the Bradyrhizobium geno sp. SA-3 Rp7b and B. zhanjiangense, respectively, whilst strain BLXBL-03 was closely related to Bradyrhizobium sp. CCBAU51745 and B. manausense BR3351. The competitiveness of these strains with Bradyrhizobium sp. SUTN9-2::GFP was analyzed, and only Bradyrhizobium sp. SMVTL-02 performed a number of occupied nodules higher than SUTN9-2::GFP. In addition, the competitiveness of the selected strain Bradyrhizobium sp. SMVTL-02 in a soil sample from the Lao PDR in the pot level was employed by tagging the SMVTL-02 with the DsRed gene. The results demonstrated that the DsRed-expressing tagged strain showed higher nodule occupancy than indigenous strains. Moreover, the results of the acetylene reduction assay (ARA), nodule number, nodule dry weight, and total plant dry weight from the pot experiment that inoculated with the SMVTL-02::DsRed were presented as having high potential to promote peanut growth as compared to non-inoculation. Thus, Bradyrhizobium sp. SMVTL-02 could be considered a potential biofertilizer inoculum for A. hypogea production in the Lao PDR